Reflecting recent work in the Dahlén and Andaloussi Groups

The delivery of oligonucleotide therapeutics, ONT, particularly small interfering RNAs, siRNAs, remains one of the foremost challenges in advancing RNA-based medicine. Traditional conjugation strategies rely on a one-to-one pairing of siRNA with a targeting ligand, such as N-acetylgalactosamine, GalNAc, which facilitates hepatocyte-specific uptake. However, this monovalent approach limits the capacity to modulate pharmacokinetics, biodistribution, and therapeutic efficiency. In this collaborative study, published in Bioconjugate Chemistry by researchers from the Dahlén group at Astra-Zeneca, Gothenburg, Sweden, and the Andaloussi team at the Karolinska Institutet, Stockholm, Sweden, the authors present a versatile peptide-based scaffold for the multimerization of siRNA, offering a structurally precise, synthetically tractable platform to improve delivery, enhance silencing potency, and reduce off-target accumulation.

Figure 1. General synthetic strategy for the siRNA multimers with the dimer-siRNA illustrated as an example. A| One-pot reaction undergoes both amide coupling between the ligand and the terminal lysine, and the SPAAC reactions between the ON and the azido-lysines core. B| After isolation of the intermediate using HPLC, the guide strands are annealed to generate the target dimer, C|.

Leveraging orthogonal bio-conjugation chemistries—including strain-promoted azide–alkyne cycloaddition, SPAAC, the researchers developed branched peptide scaffolds capable of linking two to four siRNA units to a single GalNAc moiety. This design was further extended to include linear and cyclic architectures, dual-targeting configurations, and optional endosomal escape domains. The multimeric constructs maintained excellent duplex stability, Tm ~75–77°C, despite their increased complexity, and the synthesis proceeded efficiently in aqueous media without metal catalysts.

Cell-based assays in HEK293 cells and primary human hepatocytes, PHH, demonstrated that both dimeric and tetrameric siRNA scaffolds retained potent gene silencing activity, even though the number of siRNA units quadrupled relative to the monomer. Importantly, a single GalNAc ligand was sufficient to mediate robust cellular uptake for multivalent constructs. Mechanistic studies revealed that the orientation of the siRNA conjugation, whether 3' or 5', significantly influenced activity, particularly when guide strands lacked 5' phosphorylation. Constructs with 5'-linked passenger strands outperformed their 3'-linked counterparts unless pre-activated guide strands were used, highlighting a key design parameter for multimeric siRNA efficacy.

Further functional exploration demonstrated that the peptide scaffold could successfully accommodate dual-targeting constructs, exemplified by simultaneous knockdown of PPIB and SOD1. Cyclization of the peptide scaffold, while synthetically feasible and potentially advantageous for pharmacokinetics, did not markedly affect silencing potency in vitro. Similarly, the incorporation of endosomal escape domains, EEDs, did not yield additional activity, suggesting that the balance between targeting, internalization, and release remains highly context-dependent.

Biophysical characterization revealed that the hydrodynamic diameter increased predictably with multimer size, from 2.7 nm for the monomer to 6.8 nm for the tetramer, approaching the threshold for renal filtration exclusion. Correspondingly, plasma binding studies indicated a clear increase in protein association for the larger constructs, likely due to both phosphorothioate content and overall molecular geometry.

In vivo pharmacokinetic studies in mice underscored the advantages of the multimeric design. Following intravenous administration, the tetramer displayed superior liver accumulation, improved silencing of the PPIB target gene, and a dramatic reduction in kidney exposure compared to the single-siRNA reference. Specifically, the tetramer achieved a tenfold improvement in the liver-to-kidney accumulation ratio, attributed to its size exceeding the glomerular filtration threshold. Notably, this improvement was achieved while using fourfold less GalNAc ligand per siRNA unit, offering a pathway to reduced ligand-associated toxicity.

In contrast, subcutaneous administration proved suboptimal for multimeric constructs, likely due to limited tissue diffusion and sequestration at the injection site, reinforcing the importance of administration route when deploying large, multivalent conjugates.

This study demonstrates that peptide-based scaffolds are a powerful and flexible platform for siRNA multimerization, capable of improving biodistribution, extending duration of silencing, and reducing unwanted renal clearance, all without the need for nanoparticle encapsulation. These findings not only advance the toolkit for hepatic RNA delivery but also open possibilities for targeting alternative cell types, receptors, or tissues by adjusting ligand choice and scaffold architecture.

In summary, multimeric siRNA constructs represent a compelling next-generation strategy for oligonucleotide therapeutics, offering enhanced control over pharmacological properties, improved potency, and a clear pathway toward safer, more effective RNA medicines.